phenotype correlations in aniridia

Genotype/Phenotype Correlations in Aniridia SANJOY K. GUPTA, MD, PHD, INGE DE BECKER, MD, FRCS(C), FRANC ¸ OIS TREMBLAY, PHD, DUANE L. GUERNSEY, PHD, AND PAUL E. NEUMANN, MD

● PURPOSE:

To detect and characterize mutations in cases of familial and sporadic aniridia in Maritime Canada, and to look for indications of genotype/phenotype correlation within the cohort. ● METHODS: Twelve consecutive and unrelated patients (probands) who had total or nearly complete absence of irides, and four affected relatives, were recruited from Maritime Canada. Clinical data were obtained by chart review and electroretinogram testing. Mutations in the PAX6 gene were detected by single-strand conformation polymorphism and characterized by sequence analysis. ● RESULTS: Eleven different PAX6 mutations, 10 of which are novel, were found. The four patients with congenital cataracts all had mutations in the C-terminal proline-serine-threonine (PST)–rich domain of the PAX6 protein. Electroretinograms of nine of 11 patients displayed depressed scotopic maximum response b-wave amplitudes. The greatest decrease in b-wave amplitudes was seen in patients in whom the paired domain was disrupted by mutation. ● CONCLUSION: Some aspects of the phenotype of aniridia appear to correlate with the predicted effect of point mutations on the paired and PST domains of the PAX6 protein. (Am J Ophthal-

Accepted for publication April 27, 1998. From the Division of Molecular Pathology and Molecular Genetics, Department of Pathology (Drs Gupta, Guernsey, and Neumann), Department of Ophthalmology (Drs De Becker, Tremblay, and Guernsey) and Department of Anatomy and Neurobiology (Dr Neumann), Dalhousie University, and IWK Grace Health Centre for Women and Children (Drs De Becker and Tremblay), Halifax, Nova Scotia, Canada. This work was supported by a grant from the Medical Research Council of Canada (Dr Neumann) and the IWK Grace Health Centre Research Foundation (Dr De Becker). Reprint requests to Paul E. Neumann, MD, Department of Anatomy and Neurobiology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, NS, Canada B3H 4H7; fax: (902) 494-1212; e-mail: [email protected]

0002-9394/98/$19.00 PII S0002-9394(98)00191-3

©

1998 BY

mol 1998;126:203–210. © 1998 by Elsevier Science Inc. All rights reserved.)

A

NIRIDIA (MIM 106210) IS A PANOCULAR AUTO-

somal dominant disorder in which the development of the iris, cornea, lens, angle, and retina is disturbed.1 Aniridia is typically diagnosed at birth by the bilateral absence of the iris. Vision at birth is subnormal; macular and optic nerve hypoplasia are commonly described. Congenital cataracts, typically anterior polar opacities, may also be present. Vision progressively worsens and often leads to blindness because of relentless corneal opacification, cataracts, and glaucoma. See also pp. 211–218.

Aniridia is caused by mutations affecting the PAX6 gene, located on chromosome 11p13.2–7 At the molecular level, aniridia is a haploinsufficiency disorder, caused by the loss of function of one genome copy of the PAX6 gene, which may occur through point mutations or chromosomal deletions. Inactivation of both copies of the PAX6 gene leads to a lethal phenotype.8 An important question in the clinical management of aniridia is case-to-case variability. Variability has been documented between affected individuals within families, so it is unclear how much of the variation between unrelated patients is caused by definable genotype/phenotype correlations.9,10 In other words, the degree to which differences in the location and type of PAX6 mutation influence the clinical presentation is unknown. It has been suggested that aniridia is a complete haploinsufficiency disorder in which mutations render one PAX6 allele nonfunctional or amorphic and that, therefore,

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cd/m2; 30 minutes), contact lenses (Lovac type; Medical Workshop, Groningen, The Netherlands) were set on the cornea. Cutaneous gold electrodes were fixed to the forehead (reference) and right earlobe (ground) after skin preparation. A Ganzfeld stimulator GS2000 (Nicolet, Madison, Wisconsin); produced a series of stimuli of various wavelengths to characterize rod and cone activity. Scotopic maximum responses, obtained using 1.0 cd z m22 z second flash intensity delivered at 0.2 Hz, are reported in the present study because these responses reflect the maximum output of the retina. To minimize the impact of age in our analysis of the electroretinogram signal, individual electroretinograms were normalized to age-matched normal data. DNA was prepared from EDTA-coagulated whole blood by a simple saline extraction procedure.13 Amplification of PAX6 exons (4–13 and 5A) was carried out using polymerase chain reaction (PCR) with flanking primers as described by Glaser and associates.2 Single-strand conformation polymorphism (SSCP) analysis was carried out essentially as described by Glaser and associates,2 with the following modifications. Instead of using an acrylamide-based gel, samples were electrophoresed through a 0.53 Hydrolink Mutation Detection Enhancement gel (J. T. Baker; Phillipsburg, New Jersey) using 0.53 TBE (13 TBE contains 89 mM Tris-borate, 2 mM EDTA) as electrophoresis buffer according to the manufacturer’s instructions. Single-strand conformation polymorphism gels were electrophoresed at 6 W for 12 hours (gels without glycerol) or for 17 hours (gels with 5% [v/v] glycerol) at room temperature. Gels were subsequently dried under vacuum and subjected to autoradiography. Some PCR-generated amplicons of exonic regions with identified SSCP changes were cloned into a bacterial plasmid vector (pCRII, TA cloning kit; Invitrogen, Carlsbad, California) for sequencing to identify the exact nucleotide change. To make this strategy more efficient, a second round of SSCP analysis directly from bacterial colonies was carried out to allow clear selection of allele-specific clones for sequencing.14 This procedure allows for clear selection of plasmid copies of the wild-type and mutant allele. The exact nucleotide change was determined by double-strand dideoxy sequencing.15 In the remainder of cases, PCR-generated ampli-

phenotypic variation in aniridia should be ascribed only to variability in expression of the wild-type PAX6 allele.5 Our objectives in this study were to use molecular techniques to detect and characterize PAX6 mutations in cases of familial and sporadic aniridia from Maritime Canada and to look for indications of phenotype/genotype (or clinical/molecular pathological) correlation within the cohort.

METHODS PATIENTS WERE RECRUITED WITH INFORMED CON-

sent from the Maritime Canada region (Nova Scotia, New Brunswick, and Prince Edward Island) from the practices of referring ophthalmologists. The research protocol was approved by the Ethics Review Board of the IWK Grace Health Centre, Halifax, Nova Scotia. Twelve consecutive and unrelated patients with absent or nearly absent irides were recruited; eight presented with sporadic aniridia (SPAN; patients were designated as SPAN-6, -8, -9, -10, -11, -12, -15, and -18) and four with familial aniridia (FAN; probands were designated as FAN-2A, -3, -7A, and -13A). Other family members were also invited to participate in the study. Blood from each participant (10 ml) was collected in tubes containing ethylenediaminetetraacetic acid (EDTA) for subsequent DNA preparation. Clinical data were obtained by retrospective chart review and were thus not complete in all patients. The best visual acuity reported in the documented clinical course was used in this study. Information was gathered regarding anterior segment findings (keratopathy, iris changes, cataract) and posterior segment findings (optic nerve and macular hypoplasia), and medical and surgical interventions. Patients were offered electroretinogram testing; some declined. In familial cases, an electroretinogram was performed on at least one affected individual. The electroretinographic protocol11 complied with that of the Standardization Committee of the International Society for Clinical Electrophysiology of Vision.12 Briefly, after an initial period of dark adaptation under controlled dim red illumination (2.7 3 1021 204

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TABLE 1. Clinical Information

Patient No., Sex, Age (yrs)

SPAN 9, M, 1.5 SPAN 18, F, 1.5 FAN 2A, F, 6 FAN 2B, M, 39 Father FAN 7A, F, 25 FAN 7B, F, 47 Mother FAN 7C, M, 45 Uncle SPAN 6, F, 15 SPAN 15, M, 2.5 SPAN 12, M, 11 SPAN 10, F, 6 FAN 13A, M, 17 FAN 13B, F, 49 Mother SPAN 11, M, 61 SPAN 8, F, 22 FAN 3, F, 10

Best VA RE / LE

Refractive Error RE / LE

20/270 20/270 Follows Fixates 20/160 20/160 20/100 20/100

12.0 12.0 26.0 13.0 3 90 24.0 13.0 3 90 15.50 11.0 3 90 15.50 10.75 3 90 Pl 12.0

20/80 CF NLP HM

0.50 13.0 3 90 13.75 — —

20/200 NA 20/200 NA 20/80 20/80 20/100 20/100 20/200 20/200 20/100 20/200 20/60 20/80 HM HM

11.25 12.75 3 90 11.50 13.0 3 90 15.0 11.5 3 90 16.5 120 3 90 213.5 28.50 11.25 3 90 12.75 12.25 3 90 123.0 (aphakic) Pl12.50 3 106 12.0 12.0 3 105 NA NA

20/100 20/300 20/400 20/400 20/80 20/80

13.0 2 4.50 3 180 13.0 2 1.00 3 90 210.0 11.50 3 90 26.75 14.75 10.75 3 90 16.25 10.75 3 90

Nystagmus

Keratopathy RE / LE

Iris

Cataract* RE / LE

ElectroGlaucoma retinogram Optic Nerve Macular Medical Surgical Glaucoma Hypoplasia Hypoplasia b-Wave Amp† Treatment Treatment‡ RE / LE RE / LE RE / LE Rod Cone RE / LE RE / LE

1

1/1

Absent

2/2

2/2

1/1

1/1

2

2

2/2

2/2

1

1/1

Absent

2/2

1/1

2/2

1/1

2

2

1/1

1, 2/2

1

1/1

Remnant

2/2

2/2

1/1

1/1

2

NL

2/2

2/2

1

1/1

Absent

1/2

2/2

2/2

2/2

2

2

2/2

2/2

1

1/1

Absent

1/1

2/2

NA

NA

2

NL

2/2

2/2

1

Enuc/ 1

Absent

1/1

1/1

2/2

NA

2

2

2/1

3/4, 5

1

1/1

Absent

1/1

1/1

2/2

NA

2

2

1/1

4/2

1

1/1

Absent

1/1

2/2

1/1

NA

NA

NA

2/2

6/7

1

2/2

Absent

2/2

2/2

2/2

1/1

NA

NA

2/2

2/2

1

1/1

Absent

1/1

2/2

1/1

1/1

NA

NA

2/2

2/8

1

1/1

2/2

2/2

1/1

NA

NA

2/2

2/7

1

1/1

1/1

1/1

1/1

NL

NL

1/2

9/2

1

1/1

1/1 (Congenital) Remnant 1/1 (Congenital) Absent 1/1 (Congenital)

1/1

NA

NA

NA

NA

1/1

4, 5 BE

1

1/1

Remnant

1

1/1

1

2/2

Remnant

2/2

2/2

NA

NL

NL

2/2

2/2

Absent

1/1 (Congenital) 1/1

2/2

1/1

NA

2

2

2/2

2/2

Absent

1/1

2/2

1/1

1/1

NA

NA

2/2

2/2

amp 5 amplitude; CF 5 counting fingers; Enuc 5 enucleated; HM 5 hand motion; Iris 5 slit-lamp examination; NA 5 not available; NLP 5 no light perception; Pl 5 plano; VA 5 visual acuity. *All cataract changes were acquired unless otherwise indicated. † NL 5 within normal limits of ERG b-wave amplitude: rod, 0.9 –1.1; cone, 0.85–1.15. ‡ Surgical treatment: 1 5 trabeculotomy; 2 5 corneal graft; 3 5 enucleation; 4 5 trabeculectomy; 5 5 cyclocryocoagulation; 6 5 awaiting cataract surgery; 7 5 cataract surgery; 8 5 surgery for retinal detachment; 9 5 argon laser trabeculoplasty.

cons of exonic regions with identified SSCP changes were analyzed by direct PCR sequencing using a commercially available kit (TAQCyclist; Stratagene, La Jolla, California). Sequencing ladVOL. 126, NO. 2

ders were resolved on a buffer-gradient 6% polyacrylamide sequencing gel.16 In cases in which a restriction site was created or eliminated by a mutation, the presence of the

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mutation could be verified in the proband and assessed in family members through restriction fragment length polymorphism (RFLP) analysis. Polymerase chain reaction amplifications were carried out in the presence of a radiolabeled nucleotide, as described for SSCP, in a total reaction volume of 10 ml. Restriction digests were carried out in reaction volumes of 40 ml using 10 to 20 units of the restriction enzyme according to the manufacturer’s instructions. A fraction of the restriction digest (20% to 25%) was resolved by electrophoresis in 7% to 10% polyacrylamide gels using 13 TBE as the electrophoresis buffer. Typical electrophoresis conditions were 100 v for 4 hours at room temperature for a gel 15 cm in length. Gels were subsequently dried and subjected to autoradiography.

RESULTS

FIGURE 1. Single-strand conformation polymorphism (SSCP) analysis in seven patients with aniridia. Examples of SSCP changes in three exons of the PAX6 gene are shown. Lanes 2, 9, 6, 7, 10, 11, and 13 correspond to probands FAN 2A, SPAN 9, SPAN 6, FAN 7A, SPAN 10, SPAN 11, and FAN 13A, respectively. Arrows indicate SSCP changes in exon 6 (FAN 2A and SPAN 9), exon 8 (SPAN 6 and FAN 7A), and exon 10 (SPAN 10, SPAN 11 and FAN 13A).

CLINICAL INFORMATION WAS GATHERED FOR 16 PA-

tients from the cohort (Table 1), representing the 12 probands and four affected relatives. The bestrecorded visual acuity varied from moderate (20/60 and 20/80) to hand motion/blindness. Refractive errors were hyperopic in 10 of 13 patients; exceptions were SPAN-12 (severely myopic), SPAN-8 (severely myopic), and SPAN-18, who displayed early myopia, possibly because of increased intraocular pressure. Nystagmus was present in all 16 patients. Bilateral keratopathy was a feature of 15 of 16 patients. Irides were absent in 12 of 16 patients, and four had iris remnants. Congenital cataracts were found in four of 16 patients; one of these four had required cataract extraction at age 2 years. Of the 12 remaining patients, only the four youngest had no lens opacities. Seven of 14 had optic nerve hypoplasia. Macular hypoplasia was present in seven of nine patients. Five of 16 patients had glaucoma; these five were on topical antiglaucoma medications, and all five had undergone surgical intervention for glaucoma (argon laser trabeculoplasty, trabeculectomy, trabeculotomy, or cyclocryocoagulation). There was one case of enucleation for a painful, blind eye (FAN-7B), one case of retinal detachment (SPAN-12), and three patients with cataract extraction (SPAN-6, FAN-7B, and SPAN10). 206

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Single-strand confirmation polymorphism changes, consistent with the presence of mutations or other sequence polymorphisms, were detected in 11 of 12 probands. Examples of single-strand confirmation polymorphism changes in seven probands are shown in Figure 1. An SSCP change was not detected in patient FAN-3. Using either plasmid-based or PCR-based sequencing (details in Table 2), mutations were characterized in all exons in which SSCP changes were detected (Table 2). All 11 mutations found were different and 10 were novel. The mutation (R317X) in SPAN-8 has been previously reported in a case of familial aniridia (kindred AN5).4 The novel mutation in SPAN-18 was also discovered in a sample from the same patient in another laboratory (Walter M, written communication, December 4, 1997). Of the 11 mutations, four are nonsense mutations leading to premature truncation of the open reading OF

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TABLE 2. Molecular Analysis of PAX6 Mutations With Their Predicted Products

frame (SPAN-6, -15, -12, and -8), four affect splicing (FAN-2, -7, -13 and SPAN-10), two are frameshift mutations (SPAN-9 and -11), and one is at the first Met residue, presumably leading to a N-terminal truncation of 136 amino acids (SPAN-18). In six cases, restriction recognition sites were altered by the mutation, allowing verification in the proband and molecular diagnostic testing by RFLP analyses. From the above molecular analyses, a prediction of the altered PAX6 primary amino acid structure could be made (Table 2). A prediction was also made of which domains would be affected in each case—that is, the paired domain, homeodomain, or proline-serine-threonine-rich (PST) domain (Table 2). Electroretinograms were obtained for 11 (five VOL. 126, NO. 2

sporadic aniridia patients and six members of three families). The scotopic maximum response b-wave amplitude, a reflection of the maximum retinal output, was more affected than the a-wave amplitude. Normalized b-wave amplitudes varied from 0.48 to 0.99 (Figure 2). Implicit times were generally unaffected. Results of the electroretinogram testing are described in greater detail elsewhere.11

DISCUSSION IN THIS REPORT WE DESCRIBE A COHORT OF 12 UNRE-

lated patients with aniridia. Eleven PAX6 mutations were detected in these cases, 10 of which are

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normal implicit times that suggest neural retina hypocellularity and by clinical findings of macular hypoplasia. Thirty-two other PAX6 mutations have been reported in the literature5,19 –25; however, there is little clinical information associated with most mutations. Six of the PAX6 mutations are associated with disorders other than aniridia and have been well described. These include two cases of familial Peters anomaly,24,26 one case of familial corneal opacification with minimal iris hypoplasia,27 a case of familial foveal hypoplasia,19 one case of autosomal dominant keratitis,22 and a single patient with congenital cataracts.8 Glaser and associates5 have recently classified PAX6 mutations into amorphic (that is, nonfunctional) and hypomorphic (weaker loss-of-function) alleles. Possible examples of hypomorphic alleles are those PAX6 mutations associated with a phenotype other than aniridia, such as the six cases cited above. Glaser and associates5 state that, because there is no evidence in aniridia that the mutated copy of the PAX6 gene produces a functional gene product, phenotypic variation in aniridia should be ascribed to individual variation in expression of the wild-type PAX6 allele. In other words, they suggest that PAX6 mutations leading to aniridia are amorphic PAX6 alleles. As Glaser and associates5 have noted, the lower frequency of missense mutations (amino acid substitutions) in aniridia (two of 36 cases, including the present report) than in allied disorders (three of six) is consistent with the hypothesis that aniridia alleles are amorphic and that other mutant alleles are hypomorphic. Judgments about genotype/phenotype relationships in aniridia may have been premature because most reports of PAX6 mutations lack clinical description. There are only a few reports in which detailed clinical findings of aniridia, such as we have described here, are reported with particular PAX6 mutations.3,5,8,21,28 An analysis of the clinical information of the 11 patients with defined mutations and four affected relatives has disclosed two interesting correlations to the underlying genotype (Figure 2). Patients were divided into groups defined by predictions about the status, affected (2) or unaffected (1), of the paired, homeo-, and PST domains in the mutated copy of

FIGURE 2. Genotype/phenotype correlations in aniridia. The patients were divided into groups defined by predictions about the status, affected (2) or unaffected (1), of the paired, homeo-, and PST domain in the mutated copy of the PAX6 protein. Correlations were made with respect to presence or absence of glaucoma, keratopathy, cataract (acquired or congenital), and scotopic maximum response b-wave amplitudes from electroretinograms. Absence of a dot in the table indicates that the patient does not have glaucoma, keratopathy, or cataract. Absence of a b-wave amplitude indicates that an electroretinogram was not performed. The dashed horizontal lines represent the median scotopic maximum response for each group. The box plots flanking the figure represent the twenty-fifth and seventy-fifth percentiles of normal scotopic maximum responses.

novel. These represent one quarter of the reported PAX6 mutations in human disease. We propose that PAX6 haploinsufficiency results in a reduction in the number of cells within various ocular compartments, similar to the situation in heterozygotes with PAX2 mutations, in whom optic nerve colobomas and renal hypoplasia may be caused by reduction in compartment populations.17 Heterozygotes for PAX6 mutations present with ocular conditions in which cellular differentiation does not appear to be disturbed. Nishida and associates18 have proposed that an underlying deficiency in corneal epithelial stem cells leads to progressive corneal opacification in aniridia patients. This hypothesis is also supported by electroretinogram findings of reduced b-wave amplitude with relatively 208

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the PAX6 protein. First, the four patients with congenital cataracts (SPAN-10, SPAN-11, FAN13A, and FAN-13B) all have mutations affecting exon 10, which codes for the N-terminal half of the PST domain. The three mutations described in these patients would affect the complete PST domain. The single report of a PAX6 mutation in a patient with isolated congenital cataracts describes a nonsense mutation, in exon 12, that truncates the PST domain after Ser353.8 Other PAX6 mutations affecting solely the PST domain have been reported, including one causing autosomal dominant keratitis21 and six cases of aniridia (SPAN-8 in this report and previous reports3–5,23,29); however, clinical data on these cases were insufficient either to determine the frequency of congenital cataracts in mutations affecting the PST domain or to confirm this relationship. The second possible correlation of phenotype to position and nature of the PAX6 mutation is with electroretinogram findings. The scotopic maximum response b-wave amplitude, a good measure of the total retinal activity, is reduced in a fashion that correlates with paired domain disruption (Figure 2). The lowest values of scotopic maximum response b-wave–normalized amplitudes are found in patients in whom the paired domain is disrupted by mutation (SPAN-18, FAN-2A, FAN-2B, and SPAN-9). In the two patients in whom the scotopic maximum response b-wave was near normal, the paired domain was intact (SPAN-11 and FAN-13A). Scotopic maximum response b-wave amplitudes in patients with an intact paired domain were significantly different from those in patients with a disrupted paired domain (Mann-Whitney U test; P , .05; median, 0.703 and 0.532, respectively). Our conclusion may have been influenced by the experimental design. If our sample size had been larger, perhaps we might have been able to discard the planned classification of mutations by presumed effects on the three domains and found a different way to characterize the genotype-electroretinogram correlation. Examination of Figure 2 may suggest that separation of the patients into two phenotypic classes based on electroretinogram findings has a higher concordance with division of the mutations into 59 (exons 4, 6, and 8) and 39 (exons 10 and 11) VOL. 126, NO. 2

groups. Studies of patients with other mutations may clarify the relationship. The electroretinogram is a sensitive diagnostic test of retinal function. Abnormal electroretinograms in patients with aniridia have been reported previously.30,31 Shin and associates30 also reported decreased rod and cone b-wave amplitudes; however, Mintz-Hittner and associates31 concluded that electroretinograms were not clinically useful because of large patient-to-patient variability. We also found wide variation between patients and evidence that some of that variation may be caused by genotypic differences. If genotype/phenotype correlations, such as those that we propose, exist, then all PAX6 mutations in patients with aniridia cannot be amorphic. Some may represent hypomorphic or neomorphic (for example, gain-of-function or dominant negative) alleles. PAX6 proteins with different structures have been shown to have altered biological effects. In Caenorhabditis elegans, the PAX6 gene family member vab-3 encodes a protein, containing the paired, homeo- and PST domains, which regulates neuronal specification in the head region.32 Another PAX6 gene family member in the same species, mab-18, which encodes a truncated protein lacking the paired domain, is required for specification of a peripheral sense organ in the tail region.33 In vertebrates, alternative splicing of exon 5a leads to a longer PAX6 isoform, which contains a 14 –amino acid insertion in the paired domain. A unique human ocular condition (iris hypoplasia, early cataracts, and pendular nystagmus) was found to be caused by a mutation that resulted in a relatively higher proportion of the longer PAX6 isoform.27 Molecular analysis showed that DNA binding specificity is altered by the 14 –amino acid insertion within the paired domain.27 It has also been shown that the two PAX6 isoforms bind to distinct regions in the promoter sequence of the bB2-crystallin gene.34 We propose that in some patients with aniridia, mutant PAX6 alleles may encode proteins with altered structures that have reduced function (hypomorphic) or new activities (neomorphic). Thus, in aniridia, PAX6 mutant alleles need not be complete loss-offunction (amorphic) mutants.

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16. Biggin MD, Gibson TJ, Hong GF. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci USA 1983;80:3963–3965. 17. Sanyanusin P, Schimenti LA, McNoe LA, et al. Mutation of the pax2 gene in a family with optic nerve colobomas, renal anomalies and vesiculoureteral reflux. Nat Genet 1995;9:358 –363. 18. Nishida K, Kinoshita S, Ohashi Y, Kuwayama Y, Yamamoto S. Ocular surface abnormalities in aniridia. Am J Ophthalmol 1995;120:168 –175. 19. Azuma N, Nishina S. PAX6 missense mutation in isolated foveal hypoplasia. Nat Genet 1996;13:141–142. 20. Hanson I, Brown A, van Heyningen V. A new PAX6 mutation in familial aniridia. J Med Genet 1995;32:488 – 489. 21. Martha AD, Strong LC, Ferrell RE, Saunders GF. Three novel aniridia mutations in the human PAX6 gene. Hum Mutat 1995;6:44 – 49. 22. Mirzayans F, Pearce WG, MacDonald IM, Walter MA. Mutation of the PAX6 gene in patients with autosomal dominant keratitis. Am J Hum Genet 1995;57:539 –548. 23. Sahly I, Abitbol M, Laurent C, et al. Identification of a novel PAX6 gene mutation in an aniridia patient. Hum Mutat 1996;7:377. 24. Azuma N, Nishina S, Yamada M. Missense mutation in the PAX6 gene in a family with anterior segment anomalies. ARVO abstracts. Invest Opthalmol Vis Sci 1997;38(4,suppl):S26. 25. Tang HK, Chao L-Y, Saunders GF. Functional analysis of paired box missense mutations in the PAX6 gene. Hum Mol Genet 1997;6:381–386. 26. Hanson IM, Fletcher JM, Jordan T, et al. Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters’ anomaly. Nat Genet 1994; 6:168 –173. 27. Epstein JA, Glaser T, Cai J, Jepeal L, Walton DS, Maas RL. Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing. Genes Dev 1994;8:2022–2034. 28. Martha A, Ferrell RE, Mintz-Hittner H, Lyons LA, Saunders G. Paired box mutations in familial and sporadic aniridia predicts truncated aniridia proteins. Am J Hum Genet 1994;54:801– 811. 29. Hanson IM, Seawright A, Hardman K, et al. PAX6 mutations in aniridia. Hum Mol Genet 1993;2:915–920. 30. Shin GS, Bateman JB, Heckenlively JR, Verdon WA. Electroretinographic features of aniridia. ARVO abstracts. Invest Opthamol Vis Sci 1995;36(4,suppl):S926. 31. Mintz-Hittner H, Riccardi VM, Ferrell RE, Borda RR, Justice J. Variable expressivity in autosomal dominant aniridia by clinical, electrophysiologic and diagnostic criteria. Am J Ophthalmol 1980;89:331–339. 32. Chisholm AD, Horvitz HR. Patterning of the Caenorhabditis elegans head region by the PAX-6 family member vab-3. Nature 1995;377:52–55. 33. Zhang Y, Emmons SW. Specification of sense-organ identity by a Caenorhabditis elegans Pax-6 homologue. Nature 1995;377:55–59. 34. Chambers C, Cvekl A, Sax CN, Russell P. Sequence, initial functional analysis and protein-DNA binding sites of the mouse bB2-crystallin-encoding gene. Gene 1995;166: 287–292.

ACKNOWLEDGMENTS

The authors thank the patients and their families and the referring ophthalmologists (Drs David Andrews, David Keating, Jack Quigley, Raymond LeBlanc, Robert LaRoche, Andrew Orr, Paul Rafuse, and Robert Read) for their enthusiastic participation. The authors also thank Dr Mike Walter for a gift of PAX6 PCR primers.

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